Bottom Line:
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

ABSTRACTUp to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

Figure 3: Presence of unusual Pus7-like enzymes in Sulfolobales species and A. pernix. (A) Multiple sequence alignment of Pus7 proteins from various archaeal species. Only the regions of motif IIIa, and motif II which contains the catalytic D residue, are shown. Different colours are used to highlight each of the conserved residues in motifs IIIa and II. The names of the organisms are indicated on the left, red arrows show species that contain a pre-tRNATyr-specific H/ACA sRNA. (B) 3D structure modelization of the Pab aPus7 (left) and Sso aPus7 (right) active sites. Modelization was done by using the crystal structure of M. mazei TruD (PDB 1Z2Z). Only highly conserved amino acids in the catalytic sites of RNA:Ψ-synthases are represented in the Pab and Sso aPus7 models, the K27I, R90A and H91N substitutions in Sso aPus7 can be seen.

Mentions:
As mentioned in the introduction, U to Ψ conversion at position 35 in pre-tRNATyr(GUA) is catalysed by the Pus7 stand-alone enzyme in yeast (36). By genomic sequence analysis we found putative genes for aPus7-like enzymes in the archaeal species whose genomes were completely sequenced. When the amino acid sequences of the highly conserved motifs I, II and III of RNA:Ψ-synthases were aligned for all the putative aPus7-like proteins (Figure 3A), we found a strong conservation of these motifs in the archaeal Pus7-like enzymes, except for the enzymes of the four Sulfolobales and the A. pernix species that contain putative tRNA-specific H/ACA sRNAs. These sequence divergences concern motifs II and III. Motif II contains the catalytic Asp residue (53) (Figure 3A and B). It also contains a conserved pair of Arg-His residues (54). The catalytic Asp residue is found in motifs II of the four Sulfolobales species and A. pernix, but the Arg-His pair (R78-H79 in P. abyssi) is replaced by an Ala(or Thr)-Asn (or Cys) pair (A90/N91 in S. solfataricus) (Figure 3A and B). Substitution of the Arg-His pair in motif II by other residues is also observed in a few other archaeal species that do not contain a tRNA specific H/ACA sRNA. However, in these species the highly conserved amino acids of motif IIIa are present, especially the conserved basic residue (N. maritimus and C. symbosium) which is replaced by an isoleucine in the four Sulfolobales species studied (Ile in S. solfataricus) and in A. pernix (Figure 3A). Furthermore, other amino acid substitutions are also observed in motifs II and IIIa of these five species. Altogether, these observations suggested an altered activity or specificity of the aPus7-like enzymes in S. solfataricus, S. tokodaii, S. acidocaldarius, M. sedula and A. pernix.Figure 3.

Figure 3: Presence of unusual Pus7-like enzymes in Sulfolobales species and A. pernix. (A) Multiple sequence alignment of Pus7 proteins from various archaeal species. Only the regions of motif IIIa, and motif II which contains the catalytic D residue, are shown. Different colours are used to highlight each of the conserved residues in motifs IIIa and II. The names of the organisms are indicated on the left, red arrows show species that contain a pre-tRNATyr-specific H/ACA sRNA. (B) 3D structure modelization of the Pab aPus7 (left) and Sso aPus7 (right) active sites. Modelization was done by using the crystal structure of M. mazei TruD (PDB 1Z2Z). Only highly conserved amino acids in the catalytic sites of RNA:Ψ-synthases are represented in the Pab and Sso aPus7 models, the K27I, R90A and H91N substitutions in Sso aPus7 can be seen.

Mentions:
As mentioned in the introduction, U to Ψ conversion at position 35 in pre-tRNATyr(GUA) is catalysed by the Pus7 stand-alone enzyme in yeast (36). By genomic sequence analysis we found putative genes for aPus7-like enzymes in the archaeal species whose genomes were completely sequenced. When the amino acid sequences of the highly conserved motifs I, II and III of RNA:Ψ-synthases were aligned for all the putative aPus7-like proteins (Figure 3A), we found a strong conservation of these motifs in the archaeal Pus7-like enzymes, except for the enzymes of the four Sulfolobales and the A. pernix species that contain putative tRNA-specific H/ACA sRNAs. These sequence divergences concern motifs II and III. Motif II contains the catalytic Asp residue (53) (Figure 3A and B). It also contains a conserved pair of Arg-His residues (54). The catalytic Asp residue is found in motifs II of the four Sulfolobales species and A. pernix, but the Arg-His pair (R78-H79 in P. abyssi) is replaced by an Ala(or Thr)-Asn (or Cys) pair (A90/N91 in S. solfataricus) (Figure 3A and B). Substitution of the Arg-His pair in motif II by other residues is also observed in a few other archaeal species that do not contain a tRNA specific H/ACA sRNA. However, in these species the highly conserved amino acids of motif IIIa are present, especially the conserved basic residue (N. maritimus and C. symbosium) which is replaced by an isoleucine in the four Sulfolobales species studied (Ile in S. solfataricus) and in A. pernix (Figure 3A). Furthermore, other amino acid substitutions are also observed in motifs II and IIIa of these five species. Altogether, these observations suggested an altered activity or specificity of the aPus7-like enzymes in S. solfataricus, S. tokodaii, S. acidocaldarius, M. sedula and A. pernix.Figure 3.

Bottom Line:
Up to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes.As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13.In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.

ABSTRACTUp to now, Psi formation in tRNAs was found to be catalysed by stand-alone enzymes. By computational analysis of archaeal genomes we detected putative H/ACA sRNAs, in four Sulfolobales species and in Aeropyrum pernix, that might guide Psi 35 formation in pre-tRNA(Tyr)(GUA). This modification is achieved by Pus7p in eukarya. The validity of the computational predictions was verified by in vitro reconstitution of H/ACA sRNPs using the identified Sulfolobus solfataricus H/ACA sRNA. Comparison of Pus7-like enzymes encoded by archaeal genomes revealed amino acid substitutions in motifs IIIa and II in Sulfolobales and A. pernix Pus7-like enzymes. These conserved RNA:Psi-synthase- motifs are essential for catalysis. As expected, the recombinant Pyrococcus abyssi aPus7 was fully active and acted at positions 35 and 13 and other positions in tRNAs, while the recombinant S. solfataricus aPus7 was only found to have a poor activity at position 13. We showed that the presence of an A residue 3' to the target U residue is required for P. abyssi aPus7 activity, and that this is not the case for the reconstituted S. solfataricus H/ACA sRNP. In agreement with the possible formation of Psi 35 in tRNA(Tyr)(GUA) by aPus7 in P. abyssi and by an H/ACA sRNP in S. solfataricus, the A36G mutation in the P. abyssi tRNA(Tyr)(GUA) abolished Psi 35 formation when using P. abyssi extract, whereas the A36G substitution in the S. solfataricus pre-tRNA(Tyr) did not affect Psi 35 formation in this RNA when using an S. solfataricus extract.